Anti-inflammatory molecules with tissue-targeting functions

ABSTRACT

The present disclosure provides various molecular constructs having a targeting element and an effector element. Methods for treating osteoporosis using such molecular constructs are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the divisional application of U.S. patentapplication Ser. No. 14/997,849, filed Jan. 18, 2016, which relates toand claims the benefit of U.S. Provisional Application No. 62/104,405,filed Jan. 16, 2015, U.S. Provisional Application No. 62/114,427, filedFeb. 10, 2015, and U.S. Provisional Application No. 62/137,737, filedMar. 24, 2015; the contents of the applications are incorporated hereinby reference in their entirety.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present disclosure relates to the field of pharmaceuticals; moreparticularly, to multi-functional molecular constructs, e.g., thosehaving targeting and effector elements for delivering the effector(e.g., therapeutic drug) to targeted sites.

2. Description of the Related Art

The continual advancement of a broad array of methodologies forscreening and selecting monoclonal antibodies (mAbs) for targetedantigens has helped the development of a good number of therapeuticantibodies for many diseases that were regarded as untreatable just afew years ago. According to Therapeutic Antibody Database, approximately2,800 antibodies have been studied or are being planned for studies inhuman clinical trials, and approximately 80 antibodies have beenapproved by governmental drug regulatory agencies for clinical uses. Thelarge amount of data on the therapeutic effects of antibodies hasprovided information concerning the pharmacological mechanisms howantibodies act as therapeutics.

One major pharmacologic mechanism for antibodies acting as therapeuticsis that, antibodies can neutralize or trap disease-causing mediators,which may be cytokines or immune components present in the bloodcirculation, interstitial space, or in the lymph nodes. The neutralizingactivity inhibits the interaction of the disease-causing mediators withtheir receptors. It should be noted that fusion proteins of the solublereceptors or the extracellular portions of receptors of cytokines andthe Fc portion of IgG, which act by neutralizing the cytokines or immunefactors in a similar fashion as neutralizing antibodies, have also beendeveloped as therapeutic agents.

Several therapeutic antibodies that have been approved for clinicalapplications or subjected to clinical developments mediate theirpharmacologic effects by binding to receptors, thereby blocking theinteraction of the receptors with their ligands. For those antibodydrugs, Fc-mediated mechanisms, such as antibody-dependent cellularcytotoxicity (ADCC) and complement-mediated cytolysis (CMC), are not theintended mechanisms for the antibodies.

Some therapeutic antibodies bind to certain surface antigens on targetcells and render Fc-mediated functions and other mechanisms on thetarget cells. The most important Fc-mediated mechanisms areantibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytolysis (CMC), which both will cause the lysis of the antibody-boundtarget cells. Some antibodies binding to certain cell surface antigenscan induce apoptosis of the bound target cells.

Antibodies can also serve as carriers of cytotoxic molecules or othertherapeutic agents without the antibodies' serving obvious therapeuticeffector functions. In general, those antibodies bind to“tumor-associated” antigens on target cells, but cannot cause cell lysisby themselves. Antibodies specific for CD19 and CD22 on B lymphomas arewell known. For many years, those antibodies have been explored ascarriers for cytotoxic agents, including radioactive nuclides with veryshort half-lives, such as ⁹⁰Y, ¹³¹I, and ¹⁷⁷Lu. Some antibodies havealso been studied as targeting agents for liposomes loaded withcytotoxic drugs, such as doxorubicin, paclitaxel, and amphotericin B.The field of antibody drug conjugates (ADC) has experienced an explosivephase of research and development in recent years, mainly attributing tothe development of extremely cytotoxic drugs, such as auristatin,maytansine, calicheamicin, and camptothecin, and of methodologies forconjugating the cytotoxic molecules onto antibody molecules. Those ADCshave been designed to target diffusive (or liquid) tumors of the blood,lymphoid system, and bone marrow, including various types of lymphomasand leukemia, expressing one or more unique CD markers. Some ADCs arealso being developed for solid tumors. A few of this new generation ofantibody drug conjugates have been approved for clinical uses and manyare in clinical trials.

However, in the first generation of ADCs, the cytotoxic drug moleculesare linked non-selectively to cysteine or lysine residues in theantibody, thereby resulting in a heterogeneous mixture of ADCs withdifferent numbers of drug molecules per ADC. This approach leads to somesafety and efficacy issues. For example, the first FDA-approved ADC,gemtuzumab ozogamicin, for treating acute myelogenous leukemia, is nowwithdrawn from the market due to unacceptable toxicity.

The concept and methodology for preparing antibodies with dualspecificities germinated more than three decades ago. In recent year,the advancement in recombinant antibody engineering methodologies andthe drive to develop improved medicine has stimulated the developmentbi-specific antibodies adopting a large variety of structuralconfigurations.

For example, the bi-valent or multivalent antibodies may contain two ormore antigen-binding sites. A number of methods have been reported forpreparing multivalent antibodies by covalently linking three or four Fabfragments via a connecting structure. For example, antibodies have beenengineered to express tandem three or four Fab repeats.

Several methods for producing multivalent antibodies by employingsynthetic crosslinkers to associate, chemically, different antibodies orbinding fragments have been disclosed. One approach involves chemicallycross-linking three, four, and more separately Fab fragments usingdifferent linkers. Another method to produce a construct with multipleFabs that are assembled to one-dimensional DNA scaffold was provided.Those various multivalent Ab constructs designed for binding to targetmolecules differ among one another in size, half-lives, flexibility inconformation, and ability to modulate the immune system. In view of theforegoing, several reports have been made for preparing molecularconstructs with a fixed number of effector elements or with two or moredifferent kinds of functional elements (e.g., at least one targetingelement and at least one effector element). However, it is oftendifficult to build a molecular construct with a particular combinationof the targeting and effector elements either using chemical synthesisor recombinant technology. Accordingly, there exists a need in therelated art to provide novel molecular platforms to build a moreversatile molecule suitable for covering applications in a wide range ofdiseases.

SUMMARY

The following presents a simplified summary of the disclosure in orderto provide a basic understanding to the reader. This summary is not anextensive overview of the disclosure and it does not identifykey/critical elements of the present invention or delineate the scope ofthe present invention. Its sole purpose is to present some conceptsdisclosed herein in a simplified form as a prelude to the more detaileddescription that is presented later.

In a first aspect, the present disclosure is directed to a fragmentcrystallizable (Fc)-based molecular construct that has at least onetargeting element and at least one effector element linked, directly orindirectly, to a CH2-CH3 domain of an immunoglobulin. The design of thepresent Fc-based molecular construct allows for numerous combinations ofa wide range of targeting and effector elements. Hence, the presentFc-based molecular construct may serve as a platform for constructingmulti-valent molecules.

According to certain embodiments of the present disclosure, the Fc-basedmolecular construct comprises a pair of CH2-CH3 segments of an IgG.Fc, afirst pair of effector elements, and a first pair of targeting elements.

In some embodiments, the present Fc-based molecular constructs areintended to be used in the treatment of immune diseases (in particular,autoimmune diseases) or osteoporosis. In this case, the first pair ofeffector elements consists of two effector elements, in which each ofthe two effector elements is an antibody fragment specific for tumornecrosis factor-α (TNF-α), interleukin-17 (IL-17), IL-17 receptor(IL-17R), IL-1, IL-6, IL-6R, IL-12/IL-23, B cell activating factor(BAFF), or a receptor activator of nuclear factor kappa-B ligand(RANKL); or a soluble receptor of TNF-α or IL-1. Further, the first pairof targeting elements consists of two targeting elements, in which eachof the two targeting elements is an antibody fragment specific forα-aggrecan, collagen I, collagen II, collagen III, collagen V, collagenVII, collagen IX, collagen XI, or osteonectin. In the case where thefirst pair of effector elements is linked to the N-termini of the pairof CH2-CH3 segments, the first pair of targeting elements is linked tothe C-termini of the pair of CH2-CH3 segments, and vice versa.Alternatively, when the first pair of effectors elements and the firstpair of targeting elements is both in the form of single-chain variablefragments (scFvs), then the first pair of targeting elements is linkedto the N-termini of the first pair of effector elements in a tandem ordiabody configuration, thereby forming a pair of bispecific scFvs thatare linked to the N-termini of the pair of CH2-CH3 segments.

In certain embodiments, the pair of CH2-CH3 segments is derived fromhuman IgG heavy chain γ4 or human IgG heavy chain γ1.

In some examples, the first pair of effector elements or the first pairof the targeting elements takes a Fab configuration (i.e., consisting ofthe V_(H)-CH1 domain and the V_(L)-Cκ domain); this Fab fragment islinked to the N-termini of the first and second heavy chains, so thatthe Fc-based molecular construct adopts an IgG configuration. In thesecases, the pair of elements that is not in the Fab configuration islinked to the C-termini of the pair of CH2-CH3 segments.

According to other embodiments, the Fc-based molecular construct furthercomprises a second pair of effector elements, which consists of twoadditional effector elements that are both selected from the groupdescribed above for the effector elements. According to variousembodiments, the elements of the second pair of effector elements aredifferent from those of the first pair of effector elements. In theseembodiments, the second pair of effector elements is linked to the freeC-termini of the CH2-CH3 segments.

Alternatively, the present Fc-based molecular construct furthercomprises a second pair of targeting elements, in which the twotargeting elements are both selected from the group described aboveregarding the targeting elements. According to various embodiments, theelements of the second pair of targeting elements are different fromthose of the first pair of targeting elements. In these embodiments, thesecond pair of targeting elements is linked to the free C-termini of theCH2-CH3 segments.

According to various optional embodiments, the targeting elements andeffector elements described above can be combined as desired, so as toattain the intended therapeutic effect. Some exemplary combination ofthe effector element(s) and targeting element(s) for treating immunediseases are provided in the appended claims and discussed in thedescription section bellow.

In a second aspect, the present disclosure is directed to methods fortreating various diseases. Generally, the methods involve the step ofadministrating an effective amount of the Fc-based molecular constructsaccording to the first aspect and any of the associated embodiments, toa subject in need of such treatment.

In certain embodiments, the present method is directed to the treatmentof an immune disease; in particular, an autoimmune disease.

According to some embodiments of the present disclosure, the autoimmunedisease is rheumatoid arthritis, psoriatic arthritis, or ankylosingspondylitis. In this case, the effector element is an antibody fragmentspecific for TNF-α, IL-12/IL-23, IL-1, IL-17, or IL-6, while thetargeting element may be an antibody fragment specific for collagen II,collagen IX, collagen XI, or α-aggrecan.

According to various embodiments, the autoimmune disease is psoriasis.In this case, the effector element is an antibody fragment specific forTNF-α, IL-12/IL-23, or IL-17, while the targeting element is an antibodyfragment specific for collagen I or collagen VII.

According to some other embodiments, the autoimmune disease is systemiclupus erythematosus, cutaneous lupus, or Sjögren's Syndrome. In thiscase, the effector element is an antibody fragment specific for BAFF,and the targeting element is an antibody fragment specific for collagenI, or collagen VII.

According to some embodiments, the autoimmune disease is an inflammatorybowel disease, such as Crohn's disease or ulcerative colitis. In thiscase, the effector element is an antibody fragment specific for TNF-α,and the targeting element is an antibody fragment specific for collagenIII or collagen V.

Another disease treatable by the method proposed herein is osteoporosis.According to embodiments of the present disclosure, the effector elementfor treating osteoporosis comprises an antibody fragment specific forRANKL, while the targeting element comprises an antibody fragmentspecific for collagen I or osteonectin.

BRIEF DESCRIPTION OF THE DRAWINGS

The present description will be better understood from the followingdetailed description read in light of the accompanying drawings brieflydiscussed below.

FIGS. 1A to 1F are schematic diagrams illustrating Fc-based molecularconstructs according to various embodiments of the present disclosure.

FIGS. 2A and 2B are schematic diagrams illustrating Fc-based molecularconstructs according to various embodiments of the present disclosure.

FIG. 3 shows the SDS-PAGE analysis of (scFv α CII)-(scFv αTNF-α)-hIgG4Fc.

FIGS. 4A and 4B show the ELISA results analyzing the binding of (scFv αCII)-(scFv α TNF-α)-hIgG4Fc to collagen II and TNF-α.

FIGS. 5A and 5B respectively show the SDS-PAGE and ELISA analysis of the2-chain (scFv α CII)-(scFv α TNF-α)-hIgG4.Fc-(scFv α IL-17).

FIGS. 6A and 6B respectively show the SDS-PAGE and ELISA analysis of2-chain (soluble TNF-α receptor)-IgG1.CH2-CH3-scFv α collagen II.

FIG. 7 shows the SDS-PAGE analysis of the 2-chain fusion proteincontaining intact antibody for human TNF-α and scFv specific forcollagen II.

FIGS. 8A and 8B respectively show the SDS-PAGE and ELISA analyses of the2-chain fusion protein containing intact antibody for human IL-17 andscFv specific for collagen VII.

FIGS. 9A and 9B respectively show the SDS-PAGE and ELISA analysis ofscFv α collagen VII-IgG4.CH2-CH3-scFv α BAFF.

FIGS. 10A and 10B respectively show the SDS-PAGE and ELISA analyses ofthe 2-chain fusion protein containing intact antibody for human BAFF andscFv specific for collagen VII.

FIGS. 11A and 11B respectively show the SDS-PAGE and ELISA analyses ofthe 2-chain (scFv α SPARC)-(scFv α RANKL)-hIgG4.Fc molecular construct.

FIGS. 12A and 12B respectively show the SDS-PAGE and ELISA analyses ofthe 2-chain fusion protein containing intact antibody for human RANKLand scFv specific for human osteonectin.

FIG. 13 shows the immunostaining of mouse epiphyseal bone with (scFv αCII)-(scFv α TNF-α)-hIgG4Fc and 2-chain (scFv α CII)-(scFv αTNF-α)-hIgG4.Fc-(scFv α IL-17).

FIG. 14 shows the immunostaining of mouse epiphyseal bone with 2-chain(soluble TNF-α receptor)-IgG1.CH2-CH3-scFv α collagen II.

FIG. 15 shows the bio-distribution of fluorescence-labeled (scFv αSPARC)-(scFv α RANKL)-hIgG4Fc in vivo in BALB/c mice.

In accordance with common practice, the various describedfeatures/elements are not drawn to scale but instead are drawn to bestillustrate specific features/elements relevant to the present invention.Also, like reference numerals and designations in the various drawingsare used to indicate like elements/parts, where possible.

DESCRIPTION

The detailed description provided below in connection with the appendeddrawings is intended as a description of the present examples and is notintended to represent the only forms in which the present example may beconstructed or utilized. The description sets forth the functions of theexample and the sequence of steps for constructing and operating theexample. However, the same or equivalent functions and sequences may beaccomplished by different examples.

For convenience, certain terms employed in the specification, examplesand appended claims are collected here. Unless otherwise defined herein,scientific and technical terminologies employed in the presentdisclosure shall have the meanings that are commonly understood and usedby one of ordinary skill in the art.

Unless otherwise required by context, it will be understood thatsingular terms shall include plural forms of the same and plural termsshall include the singular. Specifically, as used herein and in theclaims, the singular forms “a” and “an” include the plural referenceunless the context clearly indicated otherwise. Also, as used herein andin the claims, the terms “at least one” and “one or more” have the samemeaning and include one, two, three, or more. Furthermore, the phrases“at least one of A, B, and C”, “at least one of A, B, or C” and “atleast one of A, B and/or C,” as use throughout this specification andthe appended claims, are intended to cover A alone, B alone, C alone, Aand B together, B and C together, A and C together, as well as A, B, andC together.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in therespective testing measurements. Also, as used herein, the term “about”generally means within 10%, 5%, 1%, or 0.5% of a given value or range.Alternatively, the term “about” means within an acceptable standarderror of the mean when considered by one of ordinary skill in the art.Other than in the operating/working examples, or unless otherwiseexpressly specified, all of the numerical ranges, amounts, values andpercentages such as those for quantities of materials, durations oftimes, temperatures, operating conditions, ratios of amounts, and thelikes thereof disclosed herein should be understood as modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the present disclosureand attached claims are approximations that can vary as desired. At thevery least, each numerical parameter should at least be construed inlight of the number of reported significant digits and by applyingordinary rounding techniques. Ranges can be expressed herein as from oneendpoint to another endpoint or between two endpoints. All rangesdisclosed herein are inclusive of the endpoints, unless specifiedotherwise.

This present disclosure pertains generally to molecular constructs, inwhich each molecular construct comprises a targeting element (T) and aneffector element (E), and these molecular constructs are sometimesreferred to as “T-E molecules”, “T-E pharmaceuticals” or “T-E drugs” inthis document.

As used herein, the term “targeting element” refers to the portion of amolecular construct that directly or indirectly binds to a target ofinterest (e.g., a receptor on a cell surface or a protein in a tissue)thereby facilitates the transportation of the present molecularconstruct into the interested target. In some example, the targetingelement may direct the molecular construct to the proximity of thetarget cell. In other cases, the targeting element specifically binds toa molecule present on the target cell surface or to a second moleculethat specifically binds a molecule present on the cell surface. In somecases, the targeting element may be internalized along with the presentmolecular construct once it is bound to the interested target, hence isrelocated into the cytosol of the target cell. A targeting element maybe an antibody or a ligand for a cell surface receptor, or it may be amolecule that binds such antibody or ligand, thereby indirectlytargeting the present molecular construct to the target site (e.g., thesurface of the cell of choice). The localization of the effector(therapeutic agent) in the diseased site will be enhanced or favoredwith the present molecular constructs as compared to the therapeuticwithout a targeting function. The localization is a matter of degree orrelative proportion; it is not meant for absolute or total localizationof the effector to the diseased site.

According to the present invention, the term “effector element” refersto the portion of a molecular construct that elicits a biologicalactivity (e.g., inducing immune responses, exerting cytotoxic effectsand the like) or other functional activity (e.g., recruiting otherhapten tagged therapeutic molecules), once the molecular construct isdirected to its target site. The “effect” can be therapeutic ordiagnostic. The effector elements encompass those that bind to cellsand/or extracellular immunoregulatory factors. The effector elementcomprises agents such as proteins, nucleic acids, lipids, carbohydrates,glycopeptides, drug moieties (both small molecule drug and biologics),compounds, elements, and isotopes, and fragments thereof.

Although the terms, first, second, third, etc., may be used herein todescribe various elements, components, regions, and/or sections, theseelements (as well as components, regions, and/or sections) are not to belimited by these terms. Also, the use of such ordinal numbers does notimply a sequence or order unless clearly indicated by the context.Rather, these terms are simply used to distinguish one element fromanother. Thus, a first element, discussed below, could be termed asecond element without departing from the teachings of the exemplaryembodiments.

Here, the terms “link,” “couple,” and “conjugates” are usedinterchangeably to refer to any means of connecting two componentseither via direct linkage or via indirect linkage between twocomponents.

The term “polypeptide” as used herein refers to a polymer having atleast two amino acid residues. Typically, the polypeptide comprisesamino acid residues ranging in length from 2 to about 200 residues;preferably, 2 to 50 residues. Where an amino acid sequence is providedherein, L-, D-, or beta amino acid versions of the sequence are alsocontemplated. Polypeptides also include amino acid polymers in which oneor more amino acid residues are an artificial chemical analogue of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers. In addition, the term applies to aminoacids joined by a peptide linkage or by other, “modified linkages”(e.g., where the peptide bond is replaced by an α-ester, a β-ester, athioamide, phosphonamide, carbomate, hydroxylate, and the like.

In certain embodiments, conservative substitutions of the amino acidscomprising any of the sequences described herein are contemplated. Invarious embodiments, one, two, three, four, or five different residuesare substituted. The term “conservative substitution” is used to reflectamino acid substitutions that do not substantially alter the activity(e.g., biological or functional activity and/or specificity) of themolecule. Typically, conservative amino acid substitutions involvesubstitution one amino acid for another amino acid with similar chemicalproperties (e.g., charge or hydrophobicity). Certain conservativesubstitutions include “analog substitutions” where a standard amino acidis replaced by a non-standard (e.g., rare, synthetic, etc.) amino aciddiffering minimally from the parental residue. Amino acid analogs areconsidered to be derived synthetically from the standard amino acidswithout sufficient change to the structure of the parent, are isomers,or are metabolite precursors.

In certain embodiments, polypeptides comprising at least 80%, preferablyat least 85% or 90%, and more preferably at least 95% or 98% sequenceidentity with any of the sequences described herein are alsocontemplated.

“Percentage (%) amino acid sequence identity” with respect to thepolypeptide sequences identified herein is defined as the percentage ofpolypeptide residues in a candidate sequence that are identical with theamino acid residues in the specific polypeptide sequence, after aligningthe sequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percentage sequence identity can be achieved in variousways that are within the skill in the art, for instance, using publiclyavailable computer software such as BLAST, BLAST-2, ALIGN or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared. For purposes herein, sequence comparison between twopolypeptide sequences was carried out by computer program Blastp(protein-protein BLAST) provided online by Nation Center forBiotechnology Information (NCBI). The percentage amino acid sequenceidentity of a given polypeptide sequence A to a given polypeptidesequence B (which can alternatively be phrased as a given polypeptidesequence A that has a certain % amino acid sequence identity to a givenpolypeptide sequence B) is calculated by the formula as follows:

$\frac{X}{Y} \times 100\%$

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program BLAST in that program's alignment of Aand B, and where Y is the total number of amino acid residues in A or B,whichever is shorter.

The term “PEGylated amino acid” as used herein refers to a polyethyleneglycol (PEG) chain with one amino group and one carboxyl group.Generally, the PEGylated amino acid has the formula ofNH₂—(CH₂CH₂O)_(n)—COOH. In the present disclosure, the value of n rangesfrom 1 to 20; preferably, ranging from 2 to 12.

As used herein, the term “terminus” with respect to a polypeptide refersto an amino acid residue at the N- or C-end of the polypeptide. Withregard to a polymer, the term “terminus” refers to a constitutional unitof the polymer (e.g., the polyethylene glycol of the present disclosure)that is positioned at the end of the polymeric backbone. In the presentspecification and claims, the term “free terminus” is used to mean theterminal amino acid residue or constitutional unit is not chemicallybound to any other molecular.

The term “antigen” or “Ag” as used herein is defined as a molecule thatelicits an immune response. This immune response may involve asecretory, humoral and/or cellular antigen-specific response. In thepresent disclosure, the term “antigen” can be a protein, a polypeptide(including mutants or biologically active fragments thereof), apolysaccharide, a glycoprotein, a glycolipid, a nucleic acid, or acombination thereof.

In the present specification and claims, the term “antibody” is used inthe broadest sense and covers fully assembled antibodies, antibodyfragments that bind with antigens, such as antigen-binding fragment(Fab/Fab′), F(ab′)₂ fragment (having two antigen-binding Fab portionslinked together by disulfide bonds), variable fragment (Fv), singlechain variable fragment (scFv), bi-specific single-chain variablefragment (bi-scFv), nanobodies, unibodies and diabodies. “Antibodyfragments” comprise a portion of an intact antibody, preferably theantigen-binding region or variable region of the intact antibody.Typically, an “antibody” refers to a protein consisting of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The well-known immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Atypical immunoglobulin (antibody) structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, with each pair having one “light” chain (about 25kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of eachchain defines a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The terms variable lightchain (V_(L)) and variable heavy chain (V_(H)) refer to these light andheavy chains, respectively. According to embodiments of the presentdisclosure, the antibody fragment can be produced by modifying thenature antibody or by de novo synthesis using recombinant DNAmethodologies. In certain embodiments of the present disclosure, theantibody and/or antibody fragment can be bispecific, and can be invarious configurations. For example, bispecific antibodies may comprisetwo different antigen binding sites (variable regions). In variousembodiments, bispecific antibodies can be produced by hybridomatechnique or recombinant DNA technique. In certain embodiments,bispecific antibodies have binding specificities for at least twodifferent epitopes.

The term “specifically binds” as used herein, refers to the ability ofan antibody or an antigen-binding fragment thereof, to bind to anantigen with a dissociation constant (Kd) of no more than about 1×10⁻⁶M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M, and/orto bind to an antigen with an affinity that is at least two-foldsgreater than its affinity to a nonspecific antigen.

The term “immune disorder” as used herein refers to a disorder involvingdeficiency of humoral immunity, deficiency of cell-mediated immunity,combined immunity deficiency, unspecified immunity deficiency, andautoimmune disease.

The term “treatment” as used herein includes preventative (e.g.,prophylactic), curative or palliative treatment; and “treating” as usedherein also includes preventative (e.g., prophylactic), curative orpalliative treatment. In particular, the term “treating” as used hereinrefers to the application or administration of the present molecularconstruct or a pharmaceutical composition comprising the same to asubject, who has a medical condition a symptom associated with themedical condition, a disease or disorder secondary to the medicalcondition, or a predisposition toward the medical condition, with thepurpose to partially or completely alleviate, ameliorate, relieve, delayonset of, inhibit progression of, reduce severity of, and/or reduceincidence of one or more symptoms or features of said particulardisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease, disorder, and/orcondition, and/or to a subject who exhibits only early signs of adisease, disorder and/or condition, for the purpose of decreasing therisk of developing pathology associated with the disease, disorderand/or condition.

The term “effective amount” as used herein refers to the quantity of thepresent recombinant protein that is sufficient to yield a desiredtherapeutic response. An effective amount of an agent is not required tocure a disease or condition but will provide a treatment for a diseaseor condition such that the onset of the disease or condition is delayed,hindered or prevented, or the disease or condition symptoms areameliorated. The effective amount may be divided into one, two, or moredoses in a suitable form to be administered at one, two or more timesthroughout a designated time period. The specific effective orsufficient amount will vary with such factors as particular conditionbeing treated, the physical condition of the patient (e.g., thepatient's body mass, age, or gender), the type of subject being treated,the duration of the treatment, the nature of concurrent therapy (ifany), and the specific formulations employed and the structure of thecompounds or its derivatives. Effective amount may be expressed, forexample, as the total mass of active component (e.g., in grams,milligrams or micrograms) or a ratio of mass of active component to bodymass, e.g., as milligrams per kilogram (mg/kg).

The terms “application” and “administration” are used interchangeablyherein to mean the application of a molecular construct or apharmaceutical composition of the present invention to a subject in needof a treatment thereof.

The terms “subject” and “patient” are used interchangeably herein andare intended to mean an animal including the human species that istreatable by the molecular construct, pharmaceutical composition, and/ormethod of the present invention. The term “subject” or “patient”intended to refer to both the male and female gender unless one genderis specifically indicated. Accordingly, the term “subject” or “patient”comprises any mammal, which may benefit from the treatment method of thepresent disclosure. Examples of a “subject” or “patient” include, butare not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat,cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, thepatient is a human. The term “mammal” refers to all members of the classMammalia, including humans, primates, domestic and farm animals, such asrabbit, pig, sheep, and cattle; as well as zoo, sports or pet animals;and rodents, such as mouse and rat. The term “non-human mammal” refersto all members of the class Mammalis except human.

The present disclosure is based, at least on the construction of the T-Epharmaceuticals that can be delivered to target cells, target tissues ororgans at increased proportions relative to the blood circulation,lymphoid system, and other cells, tissues or organs. When this isachieved, the therapeutic effect of the pharmaceuticals is increased,while the scope and severity of the side effects and toxicity isdecreased. It is also possible that a therapeutic effector isadministered at a lower dosage in the form of a T-E molecule, than in aform without a targeting component. Therefore, the therapeutic effectorcan be administered at lower dosages without losing potency, whilelowering side effects and toxicity.

Diseases that can Benefit from Better Drug Targeting

Drugs used for many diseases can be improved for better efficacy andsafety, if they can be targeted to the disease sites, i.e., if they canbe localized or partitioned to the disease sites more favorably than thenormal tissues or organs. Following are primary examples of diseases, inwhich drugs can be improved if they can be preferentially distributed tothe disease sites or cells.

I Immune Disorder

According to the design of molecular constructs of the presentdisclosure, the diseases, conditions, and/or disorders treatable withthe present method is an immune disorder; for example, an autoimmunedisorder that includes, but is not limited to, psoriasis, systemic lupuserythematosus (SLE), cutaneous lupus, Sjogren's syndrome, rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, and inflammatorybowel disease.

Most of the autoimmune diseases, such as rheumatoid arthritis, systemiclupus erythematosus, Sjögren's syndrome, psoriasis, Crohn's disease,inflammatory bowel diseases, and others affect connective tissues.Regardless of the etiological nature, whether it is environmental,genetic, epigenetic, or their combinations, the affected tissues aredamaged by prolong inflammatory processes. It is rationalized in thisinvention that in bringing anti-inflammatory therapeutic agents, such asanti-TNF-α, anti-IL-17, anti-BAFF, anti-IL-6, anti-IL-12/IL-23, to thediseased connective tissues, the components of the extracellular matrixmay be employed as target antigens. The target antigens that may beconsidered include the various types of collagens, laminins, elastins,fibrillins, fibronectins, and tenascins. Connective tissues fill innearly all parts of the human body. However, due to the structural andfunctional requirements of the connective tissues in differentlocations, the types of those extracellular matrix components aredifferent, providing excellent choices for target tissue specificity.

The advantages of choosing extracellular components over cell surfaceantigens for targeting the anti-inflammatory therapeutic agents are thatthe choices of selectivity among the various types of matrix proteinsand the abundant amounts of the extracellular matrix proteins.Furthermore, because cells are not used as antigenic targets, thepotential harmful effects of direct binding to cells byanti-inflammatory agents can be avoided.

I-(i) Rheumatoid Arthritis, Psoriatic Arthritis, or AnkylosingSpondylitis

Several antibodies against TNF-α, e.g., infliximab and adalimumab, andfusion proteins of TNF-α receptor and IgG.Fc (e.g. etanercept) areapproved or in human clinical trials for use to treat rheumatoidarthritis, ankylosing spondylitis, and other autoimmune diseases. Theextracellular portion the receptor for interleukin-1 (IL-1), anakinra,is approved for treating rheumatoid arthritis. Antibodies against theshared p40 protein of IL-12 and IL-23, e.g., ustekinumab andbriakinumab, are approved for psoriatic arthritis or in trials forrheumatoid arthritis. An antibody against IL-6 receptor (tocilizumab) isapproved for rheumatoid arthritis and systemic juvenile idiopathicarthritis, and several antibodies against IL-6, e.g., sarilumab andolokizumab, are in clinical trials for treating rheumatoid arthritis. Anantibody specific for IL-17 (secukinumab) is approved for psoriasis andin clinical trials for rheumatoid arthritis and ankylosing spondylitis.

While those therapeutic agents can alleviate severe symptoms better thanpreviously available medications, they cause a range of serious sideeffects in some treated patients. For example, infliximab can causeserious blood disorders, like leukopenia and thrombocytopenia, seriousinfections, lymphoma and other solid tumors, reactivation of hepatitis Band tuberculosis, and other serious problems. Anakinra causes frequentinfections, and severe side effects on the gastrointestinal and therespiratory tracts and the blood forming organs. It is important thatthe serious side effects of these widely used therapeutic agents beminimized, while retaining or even enhancing their therapeutic effects.

In rheumatoid arthritis, joints of the knees, fingers, toes, and otherjoints are affected, and in ankylosing spondylitis, joints of the spineand the sacroiliac joint of the pelvis are affected. In the diseasedjoints, the surface of the bones and the articular cartilage lining thebone surfaces are attacked by the inflammatory immune components in thejoints. The articular cartilage in the joints is a smooth cartilage thatcontains an extracellular matrix. The cartilage is avascular andapproximately 60% of the weight is water and the remaining content iscomposed of collagens and α-aggrecan, a proteoglycan, and other matrixmolecules. Collagen II forms the major fibril in the cartilage. Aggrecanis the second most abundant component in the cartilage. Collagen XI isbound to the surface of the collagen II fibril helping to form fibrilnetworks and collagen IX is associated with collagen II and collagen XI.The cartilage has a large surface and the α-aggrecan has a structure andshape like a feather. In addition to the cartilage formation, the jointshave also ligaments, which connect adjacent bones, such as the cruciateligaments, and tendons, which connect muscles to the bones. Theligaments and tendons are formed by fibrous network of collagen types I,II, and III, and elastin and fibrillins 1 and 2.

The present invention rationalizes that the antagonist for TNF-α, IL-1,and IL-12/IL-23 can be carried to the diseased joints by using antibodyfragments, such as scFv, specific for collagen II, α-aggrecan, collagenXI or collagen IX, or alternatively, collagen I, elastin or fibrillin 1as the targeting agent. A preferred anti-collagen II antibody is onethat binds to native collagen II in the joints and does not bind toN-terminal and C-terminal propeptides, which are cleaved off duringfibril assembly. A preferred anti-aggrecan antibody is one that binds towhole native α-aggrecan molecules and does not bind to fragments thatare cleaved off and released into the blood circulation. By adopting thepresent molecular construct with scFv of anti-collagen II as targetingagent, in comparison with regular IgG against TNF-α, IL-1, andIL12/IL-23, larger proportions of the present therapeutic agents can becarried to the diseased sites and less amounts of the therapeutic agentswill be present in other irrelevant, normal tissues, especially,lymphoid organs, and hence fewer side effects will occur.

I-(ii) Psoriasis

Most patients with psoriasis or plaque psoriasis present inflammatorysymptoms primarily in the skin and not in other tissues and organs.Psoriasis involves mainly keratinocytes in part of skin in the affectedpatients. A systematic administration of monoclonal antibodiesanti-TNF-α, anti-IL-12/IL-23, and anti-IL-17 or anti-IL-17 receptor(anti-IL-17R) or other anti-inflammatory agents, such as anti-IL6,causes unwanted side effects, as discussed in the preceding section. Theserious adverse side effects of all these immune modulating antibodieshave been well documented.

A number of membrane or extracellular proteins, such as filaggrin,collagen I, which are expressed at much higher levels in the skintissues than most of other tissues, probably can be considered as thetarget proteins to shuffle therapeutic agents to the skin. Filaggrin ispresent in the tight junction between cells and is probably accessibleby antibodies in the diseased tissue sites. While collagen I is alsopresent in the bone matrix and many parts of the body, it is present inthe dermis layer of the skin in abundant proportions.

For damping the inflammatory activity caused by the diseasedkeratinocytes, which manifests psoriatic symptoms, it is not necessaryto deliver the anti-inflammatory antibody drugs to be in contact withthe keratinocytes. The keratinocytes are in the outmost, epidermis layerof the skin; blood vessels, sweat glands, and collagen fibers are in themiddle dermis layer of the skin. The inner layer is hypodermis, whereadipose tissues are. The three layers of human skin together are 2-3 mmthick. If the anti-inflammatory antibodies are delivered to the dermislayer by scFv specific for collagen I, they can diffuse into the otherlayers. Or, the antibodies can trap inflammatory cytokines in the threelayers of the skin.

Several proteins present at the dermo-epidermal junction may also beemployed as targets for carrying therapeutic agents to the skin. Theseinclude type VII collagen, type XVII collagen, and laminins type 5, 6,or 10. The dermo-epidermal junction is the area of tissue that joins theepidermal and dermal layers of the skin. The basal cells in the stratumbasale of epidermis connect to the basement membrane by the anchoringfilament of hemidesmosomes. The cells of the papillary layer of thedermis are attached to the basement membrane by anchoring fibrils, whichconsist of type VII collagen. Type XVII collagen, a transmembraneprotein (also referred to as BP180) expressed on keratinocytes, is astructural component of hemidesmosomes, multiprotein complexes at thedermal-epidermal basement membrane zone that mediate adhesion ofkeratinocytes to the underlying membrane. Laminins are structuralnon-collagenous glycoproteins present in basement membranes. Among themany types of laminins, types 5, 6, and 10 are specific of the basallamina present under stratified epithelia.

I-(iii) Systemic Lupus Erythematosus (SLE), Cutaneous Lupus, orSjogren's Syndrome

Systemic lupus erythematosus (SLE) is an autoimmune disease involvingmultiple autoantigens, such as nucleic acids, histones, and othernuclear proteins. Sjögren's syndrome is an autoimmune disease, in whichthe immune system attacks the exocrine glands, specifically the salivaryand lacrimal glands, which produce saliva and tears, respectively,resulting the symptoms of dry eyes and dry mouth, leading to infectionsand various other problems. Both of these diseases occur 9 times morefrequently in women than in men, especially in women of child-bearingages 15 to 35. SLE is a systemic autoimmune connective tissue diseaseand affects many organs and tissues. In general, those tissues andorgans, such as the heart, lungs, bladder, and kidneys, which exhibitelasticity and can expand and contract, contain collagen network. Inseveral types of SLE, cutaneous manifestation of inflammatory symptomsis prominent.

For more than 50 years, not a single new therapeutic agent had beendeveloped for SLE, until belimumab, a human monoclonal antibody specificfor BAFF was developed and approved. However, the therapeutic effect ofbelimumab for SLE has been considered to be marginal. Belimumab causes ahost of side effects, including more incidences of serious infectionsand deaths in the treatment group than the placebo group. Interestingly,in a phase II trial on Sjögren's syndrome, belimumab showed moresuccessful results than in SLE.

In addition to BAFF, researchers have been searching other therapeutictargets for SLE. While not a single inflammatory cytokine has beenidentified as mainly responsible for the pathological process in SLE,the expression of a group of genes known as downstream events of type 1interferon stimulation, which is termed “type 1 interferon signature”,has been documented in many studies. The pathogenesis of SLE has beenfound to be associated with the activation of toll-like receptors 7 and9 (TLR 7 and TLR9), which induce the expression of a group of genessimilar to that resulting from the activation by IFN-α.

Several monoclonal antibodies specific for IFN-α, includingrontalizumab, sifalimumab, and anifrolumab have been studied in clinicaltrials for the treatment of SLE. Since IFN-α is involved in manyfunctions, a systemic administration of an antibody against IFN-αwithout localized targeting to disease sites may render serious sideeffects.

I-(iv) Inflammatory Bowel Disease

Anti-TNF-α (such as adalimumab) has also been approved for treatingCrohn's disease and ulcerative colitis (a form of inflammatory boweldisease). However, as described in an earlier section, theadministration of anti-TNF-α is associated with a range of series sideeffects, including severe infectious diseases and B cell lymphoma.Therefore, in treating patients with Crohn's disease or ulcerativecolitis with anti-TNF-α, it will be desirable to distribute theadministered anti-TNF-α in favor of the intestine and colon. It has beenfound collagen III and type V are relatively abundant in the connectivetissues in the intestine and bowel.

II Osteoporosis Disease

An antibody specific for RANKL (CD254), the ligand of RANK (RANK,receptor activator of nuclear factor κ B), denosumab, is approved forthe treatment of osteoporosis. The development of denosumab represents amajor advancement in the care for osteoporosis. However, theadministration of denosumab causes common side effects, such asinfections of the urinary and respiratory tracts, cataracts,constipation, rashes, and joint pain. It is hence desirable that thetherapeutic agent is carried preferentially to the bone.

RANKL is a membrane protein, belonging to the tumor necrosis factorligand family. RANKL is detected at high levels in the lung, thymus, andlymph nodes. It is also detected at low levels in the bone marrow,stomach, peripheral blood, spleen, placenta, leukocytes, heart, thyroidand skeletal muscle. Since IgG anti-RANKL, such as denosumab, can servea therapeutic agent for osteoporosis, the molecular constructs of thisinvention should provide as better therapeutic agents than IgGanti-RANKL.

Another target for antibodies for the treatment of osteoporosis issclerostin, encoded by SOST gene. The glycoprotein is produced andsecreted by osteocytes and negatively regulates osteoblastic boneformation. The loss or defective mutation of SOST gene causesprogressive bone thickening. A defective mutation in the SOST geneincreases bone formation. Antibodies against sclerostin cause increasedbone formation, bone mineral density, and stronger bones. The phase Iand II clinical trials of two humanized monoclonal antibodies againstsclerostin, blosozumab and romosozumab, indicated that the antibodytreatment is associated with increased bone mineral density and boneformation and decreased bone resorption.

In light of the foregoing discussion, molecular platforms forconstructing the T-E molecules of this invention are provided in thepresent disclosure. Detailed discussions relating to the structure ofthe molecular construct having the “Fc” configuration are providedbelow, as well as the practical applications of each molecularconstruct.

PART I Anti-Inflammatory Molecules with Tissue-Targeting Functions

In the broad sense of the Fc-based configuration, immunoglobulinantibody can serve as the base of a targeting or effector element, andits corresponding effector or targeting element can be incorporated atthe C-terminal of its two heavy γ chains in the form of scFv domains.For a typical “Fc-based” configuration, two-chain IgG.Fc is used as thebase of the molecular platform. Each of the polypeptide chain is fusedwith one or two targeting and one or two effector elements, for a totalof two to three elements on each chain. The T-E molecule with anFc-based configuration will have a total of four to six elements (e.g.,scFv, growth factor, or cytokines). Optionally, the Fc portion of themolecular constructs also carries Fc-mediated effector functions, ADCC,and/or complement-mediated activation. While in certain otherapplications, such Fc-mediated effector functions are avoided.

In designing the Fc-based molecular constructs, targeting elements arepositioned at the N- or C-terminus. If the effector elements function bybinding to a cell surface component, such as CD3, CD16a, PD-1, PD-L1, orCTLA-4, they should also be positioned at the terminus. If the effectorelements function by binding to and neutralizing soluble factors, suchas VEGF, TNF-α, IL-17, or BAFF, they can be positioned between aterminal targeting or effector element and CH2-CH3.

In some embodiments of the present disclosure, both the effector elementand the targeting element carried by the CH2-CH3 segment (or CH2-CH3chain) are mostly comprised of amino acid residues, and for the sake ofdiscussion, these molecular constructs are referred to anti-inflammatorymolecules with tissue-targeting functions or anti-inflammatory Fc-basedmolecular construct. For example, the effector element may be anantibody fragment or a soluble receptor, while the targeting element isalso an antibody fragment. Some illustrative structures of this Fc-basedmolecular construct are discussed in this section.

Referring to FIG. 1A, which is a schematic diagram illustrating anFc-based molecular construct 800A according to certain embodiments ofthe present disclosure. As illustrated, the Fc-based molecular construct800A comprises two identical CH2-CH3 chains 810, a first pair ofeffector elements E1 linked to the N-termini of the CH2-CH3 chains 810,and a first pair of targeting elements T1 linked to the C-termini of theCH2-CH3 chains 810. In this illustrative configuration, both thetargeting element T1 and effector element E1 are antibody fragments.

In some embodiments, the CH2-CH3 chains are adopted from humanimmunoglobulins γ1 or γ4. In general, γ1 is chosen, when Fc-mediatedfunctions, such as antibody-dependent cellular cytotoxicity (ADCC) andcomplement-mediated activity (inflammatory activation or target celllysis), are desired. In the case where Fc-mediated functions areavoided, γ4 is chosen for constructing the present Fc-based molecularconstructs.

The Fc-based molecular construct 800B illustrated in FIG. 1B is quitesimilar to the Fc-based molecular construct 800A of FIG. 1A instructure, except that the two effector elements E1 are respectivelylinked to the C-termini of the CH2-CH3 chains 810, while the twotargeting effectors are respectively linked to the C-termini of theCH2-CH3 chains 810.

According to certain embodiments, both the effector elements andtargeting elements are linked to the N-termini of the CH2-CH3 chains.For example, when both the effector element and the targeting elementare in the form of single-chain variable fragments (scFvs), the effectorelement and the targeting element may be linked in a tandem or diabodyconfiguration, thereby forming a bispecific scFv that is linked to theN-terminus of the CH2-CH3 chain.

The Fc-based molecular construct 800C (FIG. 1C) comprises an Fc portion,and accordingly, each CH2-CH3 chain 810 has a T1-E1 bispecific scFvlinked to the N-terminus thereof.

As discussed above, the anti-inflammatory Fc-based molecular constructscan also use a soluble receptor (e.g., the soluble receptor of TNF-α orIL-1) as the effector element, according to certain embodiments. Inthese cases, the Fc-based molecular construct 800D (FIG. 1D) may havetwo effector elements E1 respectively linked to the N-termini of theCH2-CH3 chains 810, and two targeting elements T1 respectively linked tothe C-termini of the CH2-CH3 chains 810. It is also possible that theeffector elements and the targeting elements are respectively arrangedat the C- and N-termini of the CH2-CH3 chains; see, for example, theFc-based molecular construct 800E of FIG. 1E.

In some examples, the first pair of effector elements or the first pairof the targeting elements takes a Fab configuration (i.e., consisting ofthe V_(H)-CH1 domain and the V_(L)-Cκ domain); this Fab fragment islinked to the N-termini of the CH2-CH3 chains, so that the Fc-basedmolecular construct adopts an IgG configuration. In these cases, thepair of elements that is not in the Fab configuration may be linked tothe C-termini of the pair of CH2-CH3 segments.

For example, in the Fc-based molecular construct 800F of FIG. 1F, eachof the two targeting elements T1 comprises the V_(H)-CH1 domain 820 andthe V_(L-)Cκ domain 825, thereby forming a Fab configuration 830 that islinked to the N-termini of the CH2-CH3 chains 810, so that the Fc-basedmolecular construct 800F adopts the IgG configuration. In this case, thepair of effector elements E1 is linked to the C-termini of the pair ofCH2-CH3 chains 810.

As described above, the present Fc-based molecular construct may carry atotal of six elements at most. The additional elements may be a secondpair of effector elements or a second pair of targeting elements.

According to other embodiment, the Fc-based molecular construct 900A(FIG. 2A) comprises a second pair of targeting elements T2. In thesecases, the targeting elements T1 and T2 are linked in a tandem ordiabody configuration to form a bispecific scFv that is linked to theN-terminus of the CH2-CH3 chain 910, and the effector element E1 islinked to the C-terminus of the CH2-CH3 chain 910.

According to embodiments exemplified in FIG. 2B, the Fc-based molecularconstruct 900B comprises a second pair of effector elements E2. In thesecases, the effector element E1 and E2 are linked in a tandem or diabodyconfiguration to form a bispecific scFv that is linked to the N-terminusof the CH2-CH3 chain 910, and the targeting element T1 is linked to theC-terminus of the CH2-CH3 chain 910.

Now that the basic structural arrangements of the anti-inflammatoryFc-based molecular constructs have been discussed above, certaincombinations of particular effector element(s) and targeting element(s)are provided below for the illustration purpose.

According to some embodiments, the effector element is an scFv specificfor TNF-α, and the targeting element is an scFv specific for collagen IIor collagen IX, or α-aggrecan. According to some embodiments, each ofthe two effector elements is an scFv specific for IL-17, while thetargeting element is an scFv specific for collagen I or collagen VII.Still alternatively, each of the two effector elements is an scFvspecific for BAFF, and the targeting element is an scFv specific forcollagen I or collagen VII. In some embodiments, each of the twoeffector elements is an scFv specific for TNF-α, and the targetingelement is an scFv specific for collagen III or collagen V. In someother embodiments, the two effector elements are in the form of a Fabspecific for RANKL, and the targeting element is an scFv specific forcollagen I or osteonectin. For example, such molecular construct maytake the configuration of any of those depicted in FIGS. 1A-1C and 1F.

In some embodiments, the first pair of effector elements includes anscFv specific for TNF-α and an scFv specific for IL-17, while the firstpair of targeting elements includes an scFv specific for collagen II andan scFv specific for collagen IX. In some alternative embodiments, thefirst pair of effector elements includes an scFv specific for TNF-α andan scFv specific for IL-17, while the first pair of targeting elementsincludes an scFv specific for collagen I and an scFv specific forcollagen VII. Alternatively, each of the two effector elements is anscFv specific for BAFF, and the targeting element is an scFv specificfor collagen I or collagen VII. Still alternatively, each of the twoeffector elements is an scFv specific for RANKL, and the targetingelement is an scFv specific for collagen I or osteonectin. Thesemolecular constructs may take the configuration of any of those depictedin FIGS. 1B, 1D, and 1F.

In certain embodiments, the two effector elements are in the form of aFab antibody specific for TNF-α, while the targeting element is an scFvspecific for collagen II or collagen IX. In some embodiments, the twoeffector elements are in the form of a Fab antibody specific for IL-17,and the targeting element is an scFv specific for collagen I or collagenVII. Alternatively, the two effector elements are in the form of a Fabantibody specific for BAFF, and the targeting element is an scFvspecific for collagen I or collagen VII. Still alternatively, the twoeffector elements are in the form of a Fab antibody specific for TNF-α,and the targeting element is an scFv specific for collagen III orcollagen V.

The essence of this invention is the rationalization and conception ofthe specific combination or pairing of the targeting and effectorelements. The adoption of Fc-fusion configuration in the molecularconstructs is a preferred embodiment. It is conceivable for thoseskilled in the arts to link the pairs of targeting and effector elementsof this invention employing other molecular platforms, such as peptides,proteins (e.g., albumin), polysaccharides, polyethylene glycol, andother types of polymers, which serve as a structural base for attachingmultiple molecular elements.

PART II Uses of Anti-Inflammatory Molecules with Tissue-TargetingFunctions

Another aspect of the present disclosure is directed to the use of theanti-inflammatory Fc-based molecular constructs discussed above in PARTI.

As could be appreciated, the description in Part IV-(i) regarding therationales underlying the selection of suitable targeting and effectorelements is also applicable in this section. For example,anti-inflammatory Fc-based molecular constructs used for treatingvarious immune disorders contain an antibody fragment (e.g., scFv, Fab,and the like) specific for collagen II, collagen XI, or α-aggrecan usedas targeting elements and an antibody fragment (e.g., scFv, Fab, and thelike) specific for TNF-α and IL-17 as effector elements.

According to various embodiments of the present disclosure, the presenttreatment method involves the administration of a suitableanti-inflammatory Fc-based molecular construct to a subject in need ofsuch treatment. Specific examples of anti-inflammatory Fc-basedmolecular constructs for treating various immune disorders, inparticular, autoimmune diseases, are discussed below.

According to certain embodiments, the present method is used to treatrheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis. Inthese cases, each effector element of the anti-inflammatory Fc-basedmolecular construct is an antibody fragment specific for TNF-α,IL-12/IL-23, IL-1, IL-17, or IL-6, while each targeting element is anantibody fragment specific for collagen II, collagen IX, collagen XI, orα-aggrecan. For example, each effector element of the first pair ofeffector elements is an scFv specific for TNF-α, while each targetingelement of the first pair of targeting elements is an antibody fragmentspecific for collagen II. In other embodiments, the effector element isan scFv specific for TNF-α, while the targeting element is an antibodyfragment specific for collagen IX. Alternatively, the effector elementis an scFv specific for TNF-α, while the targeting element is anantibody fragment specific for α-aggrecan. According to variousembodiments, the above-mentioned anti-inflammatory Fc-based molecularconstructs may have the configuration of 800A, 800B, or 800C discussedabove.

Another anti-inflammatory Fc-based molecular construct for treatingrheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitiscomprises two effector elements that are in the form of a Fab antibodyspecific for TNF-α. In these cases, both targeting elements of the firstpair of targeting elements is scFvs specific for collagen II or scFvsspecific for collagen IX. Configurations of these Fc-based molecularconstructs are illustrated in FIG. 1F, for example.

The present methods are also applicable in the treatment of psoriasis.For example, the anti-inflammatory Fc-based molecular construct maycomprise effector elements of an antibody fragment specific for TNF-α,IL-12/IL-23, or IL-17, and targeting elements of an antibody fragmentspecific for collagen I or collagen VII. According to some embodiments,the effector elements are scFvs specific for IL-17, while the targetingelements are scFvs specific for collagen I. Alternatively, the effectorelements are scFvs specific for IL-17, while the targeting elements arescFvs specific for collagen VII. These anti-inflammatory Fc-basedmolecular constructs have the configuration of 800A, 800B, or 800Cdiscussed above.

Another anti-inflammatory Fc-based molecular construct for treatingpsoriasis comprises two effector elements that are in the form of a Fabantibody specific for IL-17. In these cases, both targeting elements ofthe first pair of targeting elements is scFvs specific for collagen I orscFvs specific for collagen VII. Configurations of these Fc-basedmolecular constructs are illustrated in FIG. 1F, for example.

Another set of diseases treatable by the present method using theanti-inflammatory Fc-based molecular constructs are systemic lupuserythematosus, cutaneous lupus, or Sjögren's Syndrome. In theseembodiments, each effector is an antibody fragment specific for BAFF,and each targeting element is an antibody fragment specific for collagenI or collagen VII. These anti-inflammatory Fc-based molecular constructsmay have the configuration illustrated in FIGS. 1A to 1C. As could beappreciated, the pair of effector elements may also take the form of aFab antibody specific for BAFF, and the pair of targeting elements maybe scFvs specific for collagen I or collagen VII, which takes theconfiguration of the molecular construct 800F illustrated in FIG. 1F.

In other embodiments, the present method is used to treat inflammatorybowel disease, such as Crohn's disease or ulcerative colitis. In thesecases, each effector is an antibody fragment specific for TNF-α, andeach targeting element is an antibody fragment specific for collagen IIIor collagen V. Configurations of these Fc-based molecular constructs areillustrated in FIGS. 1A to 1C, for example. Of course, the pair ofeffector elements may also take the form of a Fab antibody specific forTNF-α, while the pair of targeting elements may be scFvs specific forcollagen III or collagen V, thereby giving the configuration illustratedin FIG. 1F.

The present anti-inflammatory Fc-based molecular constructs are alsoapplicable in the treatment of osteoporosis. For example, the effectorelements are antibody fragments specific for RANKL, while the targetingelements are antibody fragments specific for collagen I or osteonectin.As could be appreciated, the antibody fragments specific for RANKL maybe scFvs so that the Fc-based molecular construct has the configurationillustrated in FIGS. 1A to 1C, or they may take the form of a Fab sothat the Fc-based molecular construct has the configuration illustratedin FIG. 1F.

It should be noted that above-examples are given for the purpose ofillustration, and treatments using anti-inflammatory Fc-based molecularconstructs with other T-E combinations are within the scope of thepresent disclosure.

EXPERIMENTAL EXAMPLES Example 1: Construction of Gene Segments Encoding2-Chain IgG4.Fc Fusion Protein Containing scFv Specific for HumanCollagen II and scFv Specific for TNF-α

Mouse B cell hybridoma II-II6B3 producing anti-collagen II antibody waspurchased from Developmental Studies Hybridoma Bank at the University ofIowa. Poly(A)+RNA was reverse-transcribed with a SuperScript III RT-PCRsystem (Invitrogen, Waltham, USA), and first strand cDNA wassynthesized. The V_(H) and V_(L) nucleotide and amino acid sequences ofII-116B3 had not been published. To determine the sequences of variableregions of II-II6B3, cDNA of V_(H) and V_(L) were amplified by PCR usinga set of DNA primers provided by Ig-primer Sets (Novagen, Madison, USA)per the manufacturer's instructions. The amino acid sequence of V_(H)and V_(L) of II6B3 monoclonal antibody specific for collagen type II(CII, or COL2) are described in SEQ ID NOs: 3 and 4. The sequences ofV_(L) and V_(H) of scFv specific for TNF-α were those of V_(L) and V_(H)of adalizumab.

Illustrated below is the configuration of 2-chain IgG4.Fc fusion proteinmolecular construct. The scFv1-scFv2-CH2-CH3 (human γ4) recombinantchain was configured by fusing two scFvs, one specific for humancollagen II and the other specific for human TNF-α, to the N-terminal ofCH2 domain of IgG4.Fc through a flexible hinge region. The first scFv(specific for collagen II) had an orientation of V_(L)-linker-V_(H) andthe second scFv (specific for TNF-α) was in V_(H)-linker-V_(L). TheV_(L) and V_(H) in each of the two scFv were connected by a hydrophiliclinker, GSTSGSGKPGSGEGSTKG (SEQ ID NO: 21). The two scFvs were connectedvia a flexible linker, (GGGGS)₃ (SEQ ID NO: 22). The sequence of therecombinant chain in the IgG4.Fc fusion protein molecular constructillustrated below is described in SEQ ID NO: 5.

Illustrated below is the configuration of the present 2-chain (scFv αcollagen II)-(scFv α TNF-α)-hIgG4.Fc molecular construct.

Example 2: Expression and Purification of Recombinant 2-Chain (scFv αCII)-(scFv α TNF-α)-hIgG4.Fc Fusion Protein

In this Example, the gene-encoding sequence was placed in pcDNA3expression cassette. Expi293F cells were seeded at a density of 2.0×10⁶viable cells/ml in Expi293F expression medium and maintained for 18 to24 hours prior to transfection to ensure that the cells were activelydividing at the time of transfection. On the day of transfection,7.5×10⁸ cells in 255-ml medium in a 2-liter Erlenmeyer shaker flask weretransfected by ExpiFectamine™ 293 transfection reagent. The transfectedcells were incubated at 37° C. for 16 to 18 hours post-transfection inan orbital shaker (125 rpm) and the cells were added ExpiFectamine™ 293transfection enhancer 1 and enhancer 2 to the shaker flask, andincubated for another 7 days. Culture supernatants were harvested andrecombinant 2-chain (scFv α CII)-(scFv α TNF-α)-hIgG4.Fc fusion proteinsin the media were purified using Protein A chromatography. Followingbuffer exchange to PBS, the concentration of (scFv α CII)-(scFv αTNF-α)-hIgG4Fc proteins was determined and analyzed by SDS-PAGE; see,FIG. 3; II-116B3 (lane 1) and (scFv α CII)-(scFv α TNF-α)-hIgG4Fc (lane2) were analyzed in 10% SDS-PAGE. The Fc-fusion molecular construct wasrevealed as the major band at about 80 kDa, consistent with the expectedsize.

Example 3: ELISA Analysis of the Binding of Recombinant 2-Chain (scFv αCII)-(scFv α TNF-α)-hIgG4.Fc Fusion Protein

To examine the binding ability of recombinant 2-chain (scFv α CII)-(scFvα TNF-α)-hIgG4.Fc fusion protein to type II collagen, ELISA assay wasperformed, using adalimumab and mouse parental monoclonal antibodyII-116B3 for comparison. ELISA plates were coated with 5 μg/mL of humantype II collagen (human COL2), mouse type II collagen (mouse COL2), andchicken type II collagen (chick COL2). 1D11 was a human IgG1 antibodyagainst mite allergen as an isotype control. Recombinant 2-chain (scFv αCII)-(scFv α TNF-α)-hIgG4.Fc fusion protein, purified anti-collagen IIantibody (II-116B3) and adalimumab were detected by HRP-conjugated goatanti-human IgG4.Fc, goat anti-mouse IgG.Fc, and goat anti-human IgG1.Fc,respectively. The ELISA results were summarized in FIG. 4A.

To examine the binding ability of 2-chain (scFv α CII)-(scFv αTNF-α)-hIgG4.Fc fusion protein to human TNF-α, ELISA assay wasperformed, along with adalimumab and mouse parental monoclonal antibodyII-116B3. ELISA plates were coated with 1 μg/mL of human TNF-α and 1μg/mL of human serum albumin as a control. Recombinant 2-chain (scFv αCII)-(scFv α TNF-α)-hIgG4.Fc fusion protein, purified anti-collagen IIantibody (II-II6B3) and Adalimumab were detected by HRP-conjugated goatanti-human IgG4.Fc, goat anti-mouse IgG.Fc, and goat anti-human IgG1.Fc,respectively. The results, as summarized in FIG. 4B, showed thatrecombinant 2-chain (scFv α CII)-(scFv α TNF-α)-hIgG4.Fc fusion proteindisplayed significant binding activity toward human TNF-α; HSA (humanserum albumin) was used as control.

Example 4: Preparation of 2-Chain IgG4.Fc Fusion Protein Containing scFvSpecific for Human Collagen II, scFv Specific for TNF-α and scFvSpecific for Human IL-17

The scFv1-scFv2-CH2-CH3-scFv3 (human γ4) recombinant chain wasconfigured by fusing three scFvs, in which the first one specific forhuman collagen II and the second one specific for TNF-α were fused tothe N-terminal of CH2 domain of IgG4.Fc through a flexible hinge region,while the third one specific for IL-17 was fused to the C-terminal ofCH3 domain.

The V_(H) and V_(L) of the scFv specific for collagen II were frommonoclonal antibody II-116B3; the V_(H) and V_(L) of the scFv specificfor TNF-α were from monoclonal antibody adalimumab; V_(H) and V_(L) ofthe scFv specific for IL-17 were from secukinumab. The first scFv(specific for collagen II) had an orientation of V_(L)-linker-V_(H), thesecond scFv (specific for TNF-α) was in the orientation ofV_(H)-linker-V_(L), and the third scFv (specific for IL-17) was in theorientation of V_(L)-linker-V_(H). The V_(L) and V_(H) in each of thethree scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG(SEQ ID NO: 21). The three scFv were fused via a flexible linker,(GGGGS)₃ (SEQ ID NO: 22).

The sequence of the recombinant chain in the IgG4.Fc fusion proteinmolecular construct of the construct illustrated below is shown in SEQID NO: 6. The expression of the constructed genes in Expi293F cells andthe purification of the expressed fusion protein were performed as inpreceding Examples. Characterization of the new construct was performedwith SDS-PAGE and ELISA. The antibody Secukinumab (Cosentyx) waspurchased from Chang Gung Hospital (Taipei, Taiwan); human IL-17 wasfrom Peprotech (NJ, USA). FIG. 5A shows the SDA-PAGE results, indicatingthat the recombinant chain of the new construct had a size of about 110kDa, consistent with the expected size. FIG. 5B shows the ELISA results,indicating that the present recombinant Fc-fusion protein had thebinding activity to human collagen II, human TNF-α, and human IL-17.

Illustrated below is the configuration of the present 2-chain (scFv αCII)-(scFv α TNF-α)-hIgG4.Fc-(scFv α IL-17) molecular construct.

Example 5: Preparation of 2-Chain IgG1.Fc Fusion Protein ContainingTNF-α Soluble Receptor and scFv Specific for Collagen II

The (TNF-α receptor)-CH2-CH3-scFv α collagen II (human γ1) recombinantchain was configured by fusing human TNF-α receptor, IgG1.Fc, and scFvspecific for collagen II. The sequences of TNF-α receptor and IgG1.Fcwere those of etanercept. Etanercept and scFv were fused via a flexiblelinker, (GGGGS)₃ (SEQ ID NO: 22). The sequence of the recombinant chainin the IgG4.Fc fusion protein molecular construct is shown in SEQ ID NO:7.

The expression of the constructed genes in Expi293F cells and thepurification of the expressed fusion protein were performed as in thepreceding Examples. Characterization of the new construct was performedwith SDS-PAGE and ELISA. Etanercept (Enbrel) was purchased from ChangGung Hospital (Taipei, Taiwan). FIG. 6A shows the SDS-PAGE results,indicating that the recombinant chain in the new molecular construct hada size of about 100 kDa, consistent with the expected size (anEtanercept molecule had a molecular weight of 150 kDa and one chain ofit was about 75 kDa in size; an scFv was about 27 kDa in size). FIG. 6Bshows the ELISA results, indicating the present molecular constructbound to human TNF-α, human collagen II, and mouse collagen II.

Illustrated below is the configuration of the present 2-chain (solubleTNF-α receptor)-IgG1.CH2-CH3-scFv α collagen II molecular construct.

Example 6: Preparation of 2-Chain Fusion Protein Containing IntactAntibody for Human TNF-α and scFv Specific for Collagen II

The IgG-scFv (human γ1) recombinant chain was configured by fusing theintact antibody specific for human TNF-α and scFv specific for collagenII (illustrated below). The sequences of intact antibody were those ofadalimumab. Adalimumab and scFv were fused via a flexible linker,(GGGGS)₃.

The construction of the heavy and light chains of recombinant genes wasbuilt by inserting the two genes into a pG1K expression cassette withthe multiple cloning site. To prepare the 2-chain fusion proteincontaining intact antibody for human TNF-α and scFv specific forcollagen type II, transfection of the expression vectors into Expi293Fcells was performed as in preceding Examples. The amino acid sequence ofthe heavy chain of 2-chain fusion protein containing intact antibody forhuman TNF-α and scFv specific for collagen type II is indicated in SEQID NO: 8, and the amino acid sequence of the light chain of the 2-chainfusion protein is indicated in SEQ ID NO: 9.

FIG. 7 shows the SDS-PAGE result, indicating that the heavy recombinantchain in the present extended IgG molecular construct had a size ofabout 75 kDa, consistent with the expected size.

Illustrated below is the configuration of the present anti-human TNF-αin extended IgG configuration with scFv specific for human collagen IIat the C-terminal.

Example 7: Preparation of 2-Chain Fusion Protein Containing IntactAntibody for Human IL-17 and scFv Specific for Collagen VII

The IgG-scFv (human γ1) recombinant chain was configured by fusing theintact antibody specific for human IL-17 and scFv specific for collagentype VII (SEQ ID NO: 20). The sequences of the intact antibody werethose of secukinumab. Secukinumab and the scFv were fused via a flexiblelinker, (GGGGS)₃ (SEQ ID NO: 22).

The construction of the heavy and light chains of recombinant genes wasbuilt by inserting the two genes into a pG1K expression cassette withthe multiple cloning site. To prepare the 2-chain fusion proteincontaining intact antibody for human IL-17 and scFv specific forcollagen type VII, transfection of the expression vectors into Expi293Fcells was performed as in the preceding Example. The amino acid sequenceof the heavy chain of 2-chain fusion protein containing intact antibodyfor human IL-17 and scFv specific for collagen type VII is indicated inSEQ ID NO: 10, and the amino acid sequence of the light chain of the2-chain fusion protein is indicated in SEQ ID NO: 11.

FIG. 8A shows the SDS-PAGE results indicating that the recombinant heavychain of the new molecular construct had a size of 75 kDa, consistentwith the expected size. FIG. 8B shows the ELISA results, indicating thatthe present molecular construct bound to human IL17 and human collagenVII. The relatively low binding to collagen VII was due to a lowconcentration the antigen used for coating the ELISA plates

Illustrated below is the present anti-human IL-17 in extended IgGconfiguration with scFv specific for human collagen VII at theC-terminal.

Example 8: Preparation of 2-Chain IgG4.Fc Fusion Protein Containing scFvSpecific for Human Collagen VII and scFv Specific for BAFF

cDNA of Mouse B cell hybridoma LH7.2 mAb producing anti-collagen VIIantibody was a gift from Dr. Purdie, cancer research UK skin tumorlaboratory at Queen Mary University of London. The V_(H) and V_(L)sequences of LH7.2 monoclonal antibody (SEQ ID NOs: 1 and 2) weredetermined in an earlier Example. The sequences of V_(L) and V_(H) ofscFv specific for BAFF were those of V_(L) and V_(H) of belimumab.

The scFv1-CH2-CH3-scFv2 (human γ4) recombinant chain (SEQ ID NO: 12) wasconfigured by fusing two scFv, spaced with IgG4.Fc, one specific forcollagen VII and the other specific for BAFF, to the C-terminal of CH3domain of IgG4.Fc through a flexible linker, (GGGGS)₃ (SEQ ID NO: 22)(illustrated below). The first scFv (specific for collagen VII) had anorientation of V_(L)-linker-V_(H) and the second scFv (specific forBAFF) was in V_(H)-linker-V_(L). The V_(L) and V_(H) in each of the twoscFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG (SEQ IDNO: 21).

The expression of the constructed genes in Expi293F cells and thepurification of the expressed fusion protein were performed as in anearlier Example. Characterization of the new construct was performedwith SDS-PAGE and ELISA. The antibody Belimumab (Belynsta) was purchasedfrom Chang Gung Hospital (Taipei); human BAFF was from GenScript (NJ,USA). FIG. 9A shows the SDA-PAGE results, indicating that therecombinant chain in the new molecular construct had a size of about80-90 kDa, consistent with or somewhat larger than the expected size.FIG. 9B shows the ELISA results, indicating that the new construct boundspecifically to human BAFF and human collagen VII.

Illustrated below is the configuration of the present 2-chain Fc-fusionmolecular construct with scFv α collagen VI I-IgG4.CH2-CH3-scFv α BAFF.

Example 9: Preparation of 2-Chain Fusion Protein Containing IntactAntibody for Human BAFF and scFv Specific for Collagen VII

The IgG-scFv (human γ1) recombinant chain was configured by fusing theintact antibody specific for human BAFF and scFv specific for collagentype VII. The sequences of intact antibody were those of belimumab.Belimumab and the scFv were fused via a flexible linker, (GGGGS)₃ (SEQID NO: 22).

The construction of the heavy and light chains of recombinant genes wasbuilt by inserting the two genes into a pG1K expression cassette withthe multiple cloning site. To prepare the 2-chain fusion proteincontaining intact antibody for human BAFF and scFv specific for collagentype VII, transfection of the expression vectors into Expi293F cells wasperformed as in preceding Examples.

The amino acid sequence of the heavy chain of 2-chain fusion proteincontaining intact antibody for human BAFF and scFv specific for collagentype VII is indicated in SEQ ID NO: 13, and the amino acid sequence ofthe light chain of the 2-chain fusion protein is indicated in SEQ ID NO:14. FIG. 10A shows the SDS-PAGE results, indicating that the recombinantheavy chain of the new molecular construct had a size of about 80 kDa,consistent with the expected size. FIG. 10B shows the ELISA results,indicating that the new extended IgG construct bound specifically tohuman BAFF and human collagen VII.

Illustrated below is the configuration of the present anti-human BAFFextended IgG configuration with scFv specific for human collagen VII atthe C-terminal.

Example 10: Preparation of 2-Chain IgG4.Fc Fusion Protein ContainingscFv Specific for Human Osteonectin and scFv Specific for RANKL

Mouse B cell hybridoma AON-1 producing anti-osteonectin (SPARC) antibodywas purchased from Developmental Studies Hybridoma Bank at theUniversity of Iowa. Poly(A)+RNA was reverse-transcribed with aSuperScript III RT-PCR system (Invitrogen), and the first strand cDNAwas synthesized. The V_(H) and V_(L) nucleotide and amino acid sequencesof AON-1 had not been published. To determine the sequences of variableregions of AON-1, cDNA of V_(H) and V_(L) were amplified by PCR using aset of DNA primers provided by Ig-primer Sets (Novagen) according to themanufacturer's instructions. The V_(H) and V_(L) sequences of AON-1monoclonal antibody specific for osteonectin are shown in SEQ ID NOs: 15and 16. The sequences of V_(L) and V_(H) of scFv specific for RANKL werethose of V_(L) and V_(H) of denosumab.

The scFv1-scFv2-CH2-CH3 (human γ4) recombinant chain (SEQ ID NO: 17) wasconfigured by fusing two scFv, one specific for human osteonectin andthe other specific for RANKL, to the N-terminal of CH2 domain of IgG4.Fcthrough a flexible hinge region (illustrated below). The first scFv(specific for osteonectin) had an orientation of V_(L)-linker-V_(H) andthe second scFv (specific for RANKL) was in V_(H)-linker-V_(L). TheV_(L) and V_(H) in each of the two scFv were connected by a hydrophiliclinker, GSTSGSGKPGSGEGSTKG (SEQ ID NO: 21). The two scFv were fused viaa flexible linker, (GGGGS)₃ (SEQ ID NO: 22).

The expression of the constructed genes in Expi293F cells and thepurification of the expressed fusion protein were performed as in anearlier Example. The characterization of the binding of the fusionprotein to human osteonectin and RANKL by ELISA was performed as in apreceding Example. Characterization of the new construct was performedwith SDS-PAGE and ELISA. The antibody Denosumab (Prolia) was purchasedfrom Chang Gung Hospital; human RANKL and human osteonectin (SPARC) werefrom GenScript. FIG. 11A shows the SDS-PAGE results, indicating that therecombinant chain in the new molecular construct had a size of about 80kDa. FIG. 11B shows the ELISA results, indicating that the new Fc-fusionconstruct bound specifically to human SPARC and human RANKL.

Illustrated below is the configuration of the present 2-chain (scFv αSPARC)-(scFv α RANKL)-hIgG4.Fc molecular construct.

Example 11: Preparation of 2-Chain Fusion Protein Containing IntactAntibody for Human RANKL and scFv Specific for Human Osteonectin

The IgG-scFv (human γ1) recombinant chain was configured by fusing theintact antibody specific for human RANKL and scFv specific for humanosteonectin. The sequences of intact antibody were those of denosumab.Denosumab and the scFv were fused via a flexible linker, (GGGGS)₃ (SEQID NO: 22).

The construction of the heavy and light chains of recombinant genes wasbuilt by inserting the two genes into a pG1K expression cassette withthe multiple cloning site. To prepare the 2-chain fusion proteincontaining the intact antibody for human RANKL and scFv specific forhuman osteonectin, transfection of the expression vectors into Expi293Fcells was performed as in preceding Examples.

The amino acid sequence of the heavy chain of 2-chain fusion proteincontaining intact antibody for human RANKL and the scFv specific forhuman osteonectin is indicated in SEQ ID NO: 18 and the amino acidsequence of the light chain of the 2-chain fusion protein is indicatedin SEQ ID NO: 19. FIG. 12A shows the SAS-PAGE results, indicating thatthe recombinant heavy chain in the extended IgG molecular construct hada size of about 80 kDa. FIG. 12B shows the ELISA results, indicatingthat the new Fc-fusion construct bound specifically to human SPARC andhuman RANKL.

Illustrated below is the configuration of the present anti-human RANKLin extended IgG configuration with scFv specific for human osteonectinat the C-terminal.

Example 12: Immunohistologic Chemical Analysis of 2-Chain IgG4.Fc FusionProtein Containing scFv Specific for Human Collagen II and scFv Specificfor TNF-α in Binding to Joint Cartilage

Immunohistologic analysis was performed in the histology core facilityof Genomics Research Center, Academia Sinica to examine whether themolecular construct, 2-chain (scFv α collagen II)-(scFv αTNF-α)-hIgG4.Fc (the configuration illustrated in an earlier Example),had affinity for binding to cartilage. Mouse bone and cartilage sampleswere obtained from FVB/N mice sacrificed by using CO₂. Femur connectedwith tibia and knee femoral ends were harvested and fixed with 10%neutral buffered formaldehyde at room temperature for 48 hours. Sampleswere then decalcified in 10% EDTA, pH 7.4, for 7 days, with dailyrenewal of the solution. After decalcification, samples were post-fixedin 10% neutral buffered formaldehyde at room temperature for 24 hoursand stored in 70% ethanol at 4° C. until dehydration by ASP6025 TissueProcessor (Leica) and paraffin embedding.

Safranin O staining was performed according to the protocol described inSchmitz et al., 2010. For immunostaining, 3-μm-thick sections weredeparaffinized and rehydrated using Leica AutoStainer XL, followed bystaining procedures described in the Tyramide Signal AmplificationBiotin kit (PerkinElmer). In brief, sections were quenched forendogenous peroxidase activity in 3% H₂O₂ for 15 minutes, followed byantigen retrieval with 1 mg/mL hyaluronidase (Sigma Aldrich) in 37° C.for 20 minutes and 20 μg/mL proteinase k (TOOLS) at room temperature for10 minutes. Sections were next blocked in TNB buffer of the TSA kit. Forstaining with mouse II-116B3 antibody, additional mouse IgG blockingreagent (Vector Laboratories) was added preceding TNB blocking. Bothprimary antibodies II-116B3 and (scFv α CII)-(scFv α TNF-α)-hIgG4Fc wereused at 50 μg/mL. Goat anti-mouse IgG Fc and goat anti-human IgG Fc(Jackson ImmunoResearch) were use at 1.6 μg/mL for incorporating HRP,which reacted with the subsequently added biotin-tyramide. The biotinlabels were then probed with streptavidin-HRP and chromogenicallyvisualized with diaminobenzidine substrate (BioGenex). Sections werecounterstained using hematoxylin and mounted with Leica CV5030Coverslipper.

FIGS. 13 panel A to 13 panel C showed the immunostaining of mouseepiphyseal bone with monoclonal antibodies II-116B3 (FIG. 13 panel A),adalimumab (FIG. 13 panel B), and (scFv α CII)-(scFv α TNF-α)-hIgG4Fc,i.e., the configuration illustrated in an earlier Example (FIG. 13 panelC), followed by HRP-labeled goat anti-mouse or goat anti-human secondaryantibodies and tyramide amplification. Type II collagen was revealed atepiphyseal articular cartilage (AC) and growth plate (GP) in FIG. 13panel A and FIG. 13 panel C. The results showed that the anti-collagenII monoclonal antibody II-116B3 stained the AC and GP parts prominently(FIG. 13 panel A), while adalimumab had no significant staining (FIG. 13panel B). The present construct also stained positively (FIG. 13 panelC). The positive staining originally revealed in brown color wasconverted to black/gray. The scale bar represents 250 μm.

Example 13: Immunohistologic Chemical Analysis of 2-Chain IgG1.Fc FusionProtein Containing scFv Specific for Collagen II and scFv Specific forTNF-α and scFv Specific for IL-17 in Binding to Joint Cartilage

The preparation of tissue thin sections, staining with the molecularconstruct, 2-chain (scFv α CII)-(scFv α TNF-α)-hIgG4.Fc-(scFv α IL-17)(the configuration illustrated in an earlier Example) and controls wereperformed as in the preceding Examples.

FIGS. 13 panel D to 13 panel E showed the immunostaining of collagen IIin mouse epiphyseal bone. 3-μm thick sections of the femoral end ofmouse knee were stained with monoclonal antibodies, an anti-IL17a mouseantibody (purchased from PeproTech, NJ, USA) (FIG. 13 panel D), and thepresent construct 2-chain (scFv α CII)-(scFv α TNF-α)-hIgG4.Fc-(scFv αIL-17) (FIG. 13 panel E), followed by HRP-labeled goat anti-mouse orgoat anti-human secondary antibodies and tyramide amplification. Theresults showed that anti-IL17 monoclonal antibody had no significantstaining, while the present construct had moderately positive staining.

Example 14: Immunohistologic Chemical Analysis of 2-Chain IgG1.Fc FusionProtein Containing TNF-α Soluble Receptor and scFv Specific for CollagenII in Binding to Joint Cartilage

The preparation of tissue thin sections, staining with the molecularconstruct, 2-chain (soluble TNF-α receptor)-IgG1.CH2-CH3-scFv α collagenII (the configuration illustrated in an earlier Example) and controlswere performed as in the preceding Example.

The staining procedure was the same as in preceding examples. The mouseepiphyseal bone tissue section samples were from the same batch. Theresults showed that the positive control II-116B3 stained strongly (FIG.14 panel A), etanercept had no significant staining (FIG. 14 panel B),and the present construct stained collagen II-containing components ACand GP positively (FIG. 14 panel C).

Example 15: Bio-Distribution of Recombinant 2-Chain IgG4.Fc FusionProtein Containing scFv Specific for Human Osteonectin (SPARC) and scFvSpecific for RANKL Using In Vivo Imaging System

A Dylight 680 Antibody Labeling Kit (Thermo Scientific) was used toconjugate denosumab and (scFv α SPARC)-(scFv α RANKL)-hIgG4Fc (scheme 67of Example 60), according to the manufacturer's instructions. 8 to10-week-old BALB/c mice were injected intravenously with PBS or 40 μglabeled antibodies. At various time points, mice were anaesthetized withisoflurane in O₂ and placed in the IVIS Spectrum In Vivo Imaging System(PerkinElmer) with a supine position. Fluorescent images were capturedwith ex/em=675/720, using the Living Image Software V3.2.

To investigate the targeting effect of (scFv α SPARC)-(scFv αRANKL)-hIgG4Fc, tissue distribution of antibodies in mice was compared,through observing fluorescent signals from the abdominal aspect.Fluorescent images from BALB/c mice were acquired and analyzed at 30minutes, 3 hours, and 28 hours after the administration of DyLight680-conjugated antibodies. 30 minutes after the injection, thepenetration of anti-SPARC, (scFv α SPARC)-(scFv α RANKL)-hIgG4Fc, andBoneTag into limbs was greater than that observed with denosumab. Inaddition to bladder accumulation, the distribution of denosumab,anti-SPARC, and (scFv α SPARC)-(scFv α RANKL)-hIgG4Fc was more dispersedafter 3 hours, while BoneTag was restricted to the head and the limbs.Bone structures were clearly resolved with remaining anti-SPARC 28 hoursafter the antibody administration.

FIG. 15 showed bio-distribution of fluorescence-labeled antibodies invivo. BALB/c mice were intravenously injected with PBS (1), denosumab(2), anti-SPARC mAb (3), (scFv α SPARC)-(scFv α RANKL)-hIgG4Fc (4), andBoneTag (5). Images were captured at indicated time points using IVISSpectrum imager and analyzed with Living Image software. Spectralunmixing was performed to distinguish tissue autofluorescence fromDyLight 680 signals. The results showed that in comparison to denosumab,the present construct was distributed more similarly to anti-SPARCmonoclonal antibody at 30 minutes after treatment. At longer timepoints, the distribution was influenced by half-lives of the reagents.Anti-SPARC and denosumab were both antibodies and had similar serumhalf-lives.

It will be understood that the above description of embodiments is givenby way of example only and that various modifications may be made bythose with ordinary skill in the art. The above specification, examplesand data provide a complete description of the structure and use ofexemplary embodiments of the invention. Although various embodiments ofthe invention have been described above with a certain degree ofparticularity, or with reference to one or more individual embodiments,those with ordinary skill in the art could make numerous alterations tothe disclosed embodiments without departing from the spirit or scope ofthis invention.

What is claimed is:
 1. A method for treating osteoporosis, comprising the step of administering to a subject in need thereof an effective amount of a molecular construct that comprises, a pair of CH2-CH3 segments of an IgG.Fc; a first pair of effector elements, wherein the effector element is a receptor activator of nuclear factor kappa-B ligand (RANKL); and a first pair of targeting elements, wherein the targeting element is an antibody fragment specific for osteonectin, wherein, when the first pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, then the first pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, and vice versa, or when the first pair of effectors elements and the first pair of targeting elements are both in the form of single-chain variable fragments (scFvs), then the first pair of targeting elements is linked to the N-termini of the first pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments.
 2. The method of claim 1, wherein the pair of CH2-CH3 segments is derived from human γ4 or γ1 immunoglobulin.
 3. The method of claim 1, wherein when the first pair of effector elements is in the form of an antigen-binding fragment (Fab), and the first pair of targeting elements is in the form of scFvs, and vice versa; then the Fab and scFvs are respectively linked to the N-termini and C-termini of the CH2-CH3 segments, so that molecular construct adopts an extended IgG configuration.
 4. The method of claim 1, wherein, the effector element is an scFv specific for RANKL; and the targeting element is an scFv specific for osteonectin.
 5. The method of claim 1, wherein, the two effector elements are in the form of a Fab specific for RANKL; and the targeting element is an scFv specific for osteonectin.
 6. A molecular construct, comprises, a pair of CH2-CH3 segments of an IgG.Fc; a first pair of effector elements, wherein the effector element is a receptor activator of nuclear factor kappa-B ligand (RANKL); and a first pair of targeting elements, wherein the targeting element is an antibody fragment specific for osteonectin, wherein, when the first pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, then the first pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, and vice versa, or when the first pair of effectors elements and the first pair of targeting elements are both in the form of single-chain variable fragments (scFvs), then the first pair of targeting elements is linked to the N-termini of the first pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments.
 7. The molecular construct of claim 6, wherein the pair of CH2-CH3 segments is derived from human γ4 or γ1 immunoglobulin.
 8. The molecular construct of claim 6, wherein when the first pair of effector elements is in the form of an antigen-binding fragment (Fab), and the first pair of targeting elements is in the form of scFvs, and vice versa; then the Fab and scFvs are respectively linked to the N-termini and C-termini of the CH2-CH3 segments, so that molecular construct adopts an extended IgG configuration.
 9. The molecular construct of claim 6, wherein, the effector element is an scFv specific for RANKL; and the targeting element is an scFv specific for osteonectin.
 10. The molecular construct of claim 6, wherein, the two effector elements are in the form of a Fab specific for RANKL; and the targeting element is an scFv specific for osteonectin. 